Abstract Enteroaggregative E. coli (EAEC) is an important pathogen of traveler's diarrhea, diarrhea in industrialized countries and growth faltering in developing countries. In this application, we propose to study EAEC pathogenesis in the human intestinal mucosa through the use of newly established human enteroid/colonoid model, which is an ex vivo self?propagating human intestinal primary culture. This application comprises a consortium among investigators at Johns Hopkins University (JHU), who have pioneered the development of the enteroid/colonoid model, and researchers at the University of Maryland (UMD) and the University of Virginia, who are leaders in the study of the pathogenesis of diarrheal disease cause by diarrhegenic E. coli and Shigella. This project will leverage the existence of the new colonoid monolayer model. We will focus on characterization of EAEC interactions with colonoid and colonoids/leukocyte models, in particular to dissect the roles of AAFs, which have emerged as the principle EAEC virulence factor in multiple models, and the function of SPATEs, which have shown broad spectrum activities on mucin and leukocyte glycoproteins, promoting immunomodulatory effects. This project will comprise two Specific Aims. In Aim 1, we will characterize interactions of EAEC with the intestinal mucosa. We will extend ongoing studies which suggest that EAEC binding to MUC glycoproteins is fundamental to its pathogenesis. We will extend structure-function studies of the AAF adhesin and illuminate the contribution of AAF basic residues to interaction of EAEC with the human intestinal mucosa. We will determine the contribution of AAF mucin-binding residues to EAEC interaction with the human mucosa. We will characterize the contributions of Pic and dispersin to AAF-mediated interaction with the mucosa using time-lapse microscopy methods. In Aim 2 we will characterize the immunomodulatory role of class 2 SPATEs on the intestinal mucosa. We will address the potential roles of cleavage of intestinal glycoproteins in EAEC pathogenesis. We will dissect the inflammatory response through binding and cleavage of O-linked glycoproteins such as the MUC signaling proteins on the intestinal mucosa. We will also characterize the immunomodulatory effect of Pic through cleavage of inflammatory cytokine receptors in the colonoid/macrophage system. The work described here will continue to advance the general study of EAEC pathogenesis, generating fundamental insights that will illuminate aspects of pathogenesis relevant to other enteric pathogens. We will work closely and synergistically with investigators in other projects and the Core components.